BOBOIR999999X @x  /-Z Jeremy BrownxHHB@J {hhx Jl2 JGP'd S600PZ1/Q9k/Kk/KH29Q 4eDSETyz(,8@<,(0 "3 !3,b-3:83EL3 P3 [1 d3 l(2 |vP P `  01 2 3 4 yz{ | } ~ L]Z[opMNno       S T        @        &'NOHI !"GH!!!89: y''''ghqr!Y!Z""""& & &&(n(o))**++-3-4-------....1$1%2)2*447>7?7@7c7d:7:8:G:H:d:e<8<9>>@@AA_AAB,BeBBBC#CZ(C[(C\(C(C(F#(F$(F(F(H(H (I*(I+IEIFKKMMOOOP0PuPPQ6QqQQQR+-R,-T(T(Tl(Tm(V'(V((V(V-W-W-XXXXZZ\T-\U-\-\-^w-^x-_-_-_-_-a aaa%a&b(b(c(c(c(c(d-dddddffffffgjkjlllmmooo7o8pWpXpps s tZt[ttuhuivMvNvOvPvevfxxyy y y y y z z zzL/ 0 1=4 gExy { ;~    C C F    6   Q : M N  m o     t        $ ' )   O Q       I K       ! 9" F H g h " , "8 : #> &y + . +o 1r +   + 1 +  1 +g h - - -  - .  . 3. 7@ %7b :7 :8  :F :H 4:c  :d :e : $: =S $=X CZ *I+ 4IE IF /I )I /L$ )L) /R+ 0T *V 0X X 4X 3X YP $YU \T 0a a 5a 4a& /a )a /b *d 0d d d  d e  e f  f f  f 3f gc  gi h  h j  j l 3l o 3o6 pX 3p s  s   s t[  t t t u& u. uh 8u vP  vd vf xw :x y yN :y y y y y z z z 4Z0D, 9=( v=$ v$4 +  #6-&cx(f+A.1 472:L@CG4IFK1NMiQTpuWZe^aeim3ptvEInclusion Counter Manual v1.0 Jeremy Brown 1998 Inclusion Counter , Object-Image and NIH Image are completely free! Table of Contents 1. About NIH Image 2. About Inclusion Counter v1.0 3. Registration 4. Installation 5. Acquisition and storage of Images 6. How to process images using Inclusion Counter v1.0 7. Macro files 8. Photoshop 4.0 and Inclusion Counter 10. Acknowledgements 1. About NIH Image NIH Image is a public domain image processing and analysis program for the Macintosh. It was developed at the Research Services Branch (RSB) of the National Institute of Mental Health (NIMH), part of the National Institutes of HZealth (NIH). If you do not already have a copy of NIH Image you may freely download the latest version from the following location: http://rsb.info.nih.gov/nih-image/download.html or obtain it on floppy disk from the national Technical Information Service, Springfield, Virginia, part number PB95-500195GEI. A free PC version of Image, called Scion Image for Windows, is available from Scion Corporation. There is also Image/J, a Java program inspired by Image that "runs anywhere". However, Inclusion counter currently only runs on NIH Image for the Macintosh and I have no plans to port it to Scion Image for Windows. I will however port inclusion counter to Image/J as soon as it supports macros. System Requirements Image requires a colour capable Macintosh and at least 4 MB of free RAM. 32 MB or more of RAM is recommended for working with 3D imagDes, 24-bit colour or animation sequences. System 7.0 or later is required. Image directly supports, or is compatible with, large monitors, flatbed scanners, film recorders, graphics tablets, Postscript laser printers, photo typesetters and colour printers. More information about NIH Image can be found at: http://rsb.info.nih.gov/nih-image/ 2. About Inclusion Counter v1.0 Inclusion Counter is a suite of NIH Image macros designed specifically for the automated enumeration and measurement of Chlamydial inclusions in cell culture and tissue sections. It will also count and measure just about any other type of particulate object. I hope that even if you have no previous experience of NIH Image you will find these macros both easy to use and of significant use in your research. Inclusion Counter was originally designed for use= with NIH Image 1.61. However, it also works without problems in Norbert Vischers Object-Image 1.62, an enhanced version of NIH Image with some really good additional features. 3. Registration I would be very grateful if you would register your copy of Inclusion counter. Why register Inclusion Counter? Well, why not? If you register you will receive software updates and free advice. Inclusion Counter v1.0 will be the only version of Inclusion counter distributed freely over the internet. All future versions will be sent directly to registered users or in response to request. Inclusion counter is and will remain free. The purpose of registration is really so that you can give me feedback on Inclusion Counter allowing me to tailor future versions of inclusion counter to suit your needs. Finally, Ive put a l=ot of hard work into Inclusion Counter and I would like to know who is using it. To register your copy: email me at: brownj@netmatters.co.uk. Under "Subject" type "Register Inclusion Counter". In the "Body" of the email simply give details of where and what you are using Inclusion Counter for. Many thanks in advance. Jeremy Brown Moredun Research Institute 408 Gilmerton Road Scotland UK EH17 7JN 4. Installation 1. Drag the "ROI Folder" supplied with Inclusion Counter into the folder called "Preferences" in your "System Folder". 2. In the "Inclusion Counter Folder" double Click on the "Help With Paths" icon, this will automatically launch NIH Image and display a text window called "Help With Paths". 3. Choose "Load Macros from Window" from the "special" menu. Then choose "Help With Paths" from the "special" menu, and follow the on screen instructions. 4. "Help With Paths" will create a text file called "Inclusion Counter Help File". You will be prompted to save this file, you may also find it useful to print a copy. 5. The "Inclusion Counter Help File" should display the correct Path Name for "ROI" at the bottom of the window. 6. Follow the instructions in the "Inclusion Counter Help File". 7. Finally, discard the original file called "ROI" in the "ROI Folder". 8. Inclusion Counter should now be ready to work! NB: If you move the location of the "ROI folder" after you have run "help with paths" INCLUSION COUNTER WILL NOT WORK! Keep a copy of the original self extracting archive in case you need to repeat the installation process. Should you have any problems email me at brownj@Netmatters.co.uk with the Subject Heading Inclusion Counter Problems. 5. Acquisition and storage of Images For acquisition products go to: http://www.meyerinst.com/html/mi_proda.htm Inclusion Counter does not include any facilities for image capture. This is mainly because I capture all my images using different software. For example, the Mac I most commonly capture images on uses a piece of commercial software called ColourVision which I prefer to NIH-Image for acquisition purposes. I also, on occasion use a PC for acquiring images. It simply does not matter how you acquire your images! However, if you do not use NIH-Image for acquisition make sure that the software you are using saves in a format that NIH-Image recognises. The two major Image Formats supported by NIH-Image are listed below in order of preference of use with Inclusion Counter. TIFF: Tag Image File Format; suffix .tif on wintel based PCs. This is one of the most widely used image formats andv is the format you need to use if you are processing colour images in NIH-Image. NIH Image will open a colour TIFF file as a stack of 3 images, one for each colour channel Red, Green and Blue (RGB). If you cannot save in TIFF format in the application you are using to capture images (very unlikely, but beware of manufacturers who seek to enslave you to their own weird image formats!) convert the images into TIFF format using an application such as Photoshop or GraphicConverter. Do not ever consider capturing images in JPEG format. JPEG is bad news for image analysis purposes. PICT: Macintosh Picture. Again this is a widely used Image format, indeed it is the standard image format for Macintosh compatible computers. However, NIH-Image will open a colour PICT file as a grey scale image not as a RGB stack. You should only use PICT if you are using a Black & White camera or do not need colour channel separation. Convert colour PICT format Images to TIFF for use in NIH Image using an application such as Photoshop 4.0 or GraphicConverter, see above. Both of these applications have the ability to automate the conversion of whole folders of files from PICT to TIFF, not to mention a huge number of other formats as well. I have included a Photoshop action to batch convert PICT to TIFF with inclusion counter in the Goodies folder. If you do not have Photoshop, GraphicConverter is share ware and is available from all manner of sources, price $30. This brings me to storage. If you, like I, do not have your own Macintosh linked in some way to a microscope and digital camera you are going t4o need removable media to store your images on. In fact it would be fairly unrealistic to assume that you will not need some form of removable storage media even if you are directly linked up to your acquisition setup or indirectly by a network. Which type of media you choose from the bewildering array which is currently available is up to you, but you must take into consideration a number of things. 1. Speed and size: a typical high resolution colour TIFF image takes up about 1.1 MB of space and will take a couple of seconds to save on even the fastest removable media. Do not even consider saving you images on floppy disks! 2. Portability: Obviously you will need the same type of storage media at both ends or an external device which you can easily move between machines, I use Jaz Drives. If the machine yo u are going to capture images on already has some kind of removable storage use that, if you can. However consider this, if you want to take several hundred images, I take up to 480 per 96 well plate, you really want media with a capacity greater than 500 MB and with speed compatible to a hard disk. 3. Compatibility: SCSI compatible drives are the best option. All Macs support external SCSI drives, due to cost PCs do not. If you are going to but one drive to move between Mac and PC bear in-mind you may have to buy a SCSI card for the PC. Newer 100 MB zip drives have both SCSI and PC style Parallel ports. Parallel ports are slower but so are zip drives! The best options available: Iomega Jaz Drives I and II: take 0.5 or 1 GB and 0.5, 1 or 2 GB disks respectively- and are very fast. Media cost about /$ 60 for 1 GB and /$ 120 for 2 GB. Can be written in Mac or PC format and has a life time guarantee. However PC-formatted Jaz Disks can behave strangely if you save Mac Data to them. So (unusually since the ability to read most PC media is built into the Mac OS) if you are transferring data PC-Mac, format the disk in your Mac and buy one of the very reasonably price PC utilities which allow you to read Mac formatted media, format the disk in your Mac and buy one of the very reasonably price PC utilities which allow you to read and write Mac formatted media, e.g DataViz Conversions Plus (long file names preserved). This problem should no longer exist in Mac OS 8 and OS 8.1. The 1.5 GB Syquest Syjet which is slightly faster than Jaz I slower than Jaz II. Media xcost about /$ 60. Use PC formatted Disks when transferring data between Mac and PC. The 750 MB Noma Drive is slightly faster and cheaper than the Jaz or Syjet drives and media for these drives is very cheap, about /$ 30. This drive would be my choice if I, like so many people had not already bought a Jaz drive! If you want a drive to move between computers an external SCSI version would be Ideal. I dont know about formatting for these drives but I guess PC-formatted versions of this media will work in the Mac. Magneto Optical drives which vary from 230 MB up to 2.6 GB and are slower than any of the above. Media tenfds to be cheaper than Jaz or Syquest, although the drives themselves can be very expensive. Possibly the most robust storage available, although some would tell you differently. Recordable CD-Rom drives, much slower than Jaz or Syquest and can only be written to once. However, media is very cheap and provides a stable and permanent record of your work. They can also be written in a variety of formats recognised by both Mac and PC. More suited to archiving images and Data. Re-Recordable CD-Roms, not only do these new drives do the job of CD-Rom recorders above but they can also use a more expensive form of media (10x the price!) which can be written to repeatedly. As with recordable CD-Rom drives, a bit slow for ever day use but a good candidate for archiving images and Data. There are also some new drives promised in the near future: Orb 2.1 GB bigger, faster and cheaper than Jaz or Syquest (http://www.castlewoodsystems.com) and a 1.0 GB Syquest drive which is already here, but as always with Syquest a slightly different format from previous Syquest drives. Looks pretty good though. My own preference is currently a combination of Jaz and recordable CD-Rom. 6. How to process images using Inclusion Counter v1.0 Once you have Installed Inclusion Counter using the macro Help With Paths you can get down to business. I hope that I have designed Inclusion Counter to be as intuitive as possible. However, I appreciate that it may daunting at first. Which macro should you use? Batch Count Inclusions v1.0 and Manual Density Slice v1.0 essentially do the same thing. Batch Count Inclusions v1.0 is by far the faster of the two. The main difference is that Manual Density Slice v1.0, as its name may suggest to you allows the density slice of each image to be set manually by the user. Whilst this is time consuming compared to Batch Count Inclusions v1.0 which uses a single density slice setting for the whole batch of images to be processed, it does allow the user greater flexibility. For example, Manual Density Slice v1.0 is better suited to tissue section work. In cell culture I find that Manual Density Slice v1.0 offers very little advantage over Batch Count Inclusions v1.0. Indeed, statistically it seems to make little difference to the accuracy of Inclusi on Counter. However you may wish to experiment with it. A third macro No Background Subtraction v1.0 does almost exactly the same thing as Manual Density Slice v1.0. However, as the name may suggest to you it does not do a 2D rolling ball background subtraction. In most cases I would strongly advise you to use the other 2 macros, particularly if you are working with 96 well plates or similarly subjects of poor optical quality. Unless you are using a 604e MACH5 or G3 equipped Mac No Background Subtraction v1.0 will be much faster than Batch Count Inclusions v1.0. You can use No Background Subtraction v1.0 for cytospins and chamber slides of non-confluent cells. Photoshop Helper v1.0 is designed to work in conjunction with the Photoshop 4.0 actions palette Inclusion Counter Actions supplied with Inclusion Counter. I use it for quality control purposes. Basically Photoshop Helper v1.0 counts and measures binary outlines of inclusions made manually in Photoshop 4.0. For obvious reasons, If you do not have Photoshop 4.0 it is not much use! Whilst I have tested the accuracy of Inclusion Counter extensively for my own use, I recommend that you use Photoshop Helper v1.0 to check the accuracy of Inclusion Counter in each particular cell type and staining system you are using. See Notes on2 Photoshop later on in this manual. Which colour channel should I use? Use the rectangular selection tool to out line one or two inclusions and some typical cellular background. The select [C] Choose a Colour Channel from the Special menu and let Inclusion Counter show you the best combination of colour channels to use. For example, red inclusions on a blue background show up best in either the green channel or a combination of the blue and green channels. Try and place your selection so that the object of interest is in the centre of the selection. Inclusions Counter will do a surface plot along the diagonal running the top left to the bottom right corners of the selection to give you some idea of the difference in grey values between the object of interest and the background. 7. Macro files Batch Count Inclusions v1.0 1. Set the region of the images which you are interested in or ROI. Choose any of the area selection tools (FIG 1)and outline the area you want. If like me your camera is quite old and has a few bad lines at one edge you can eliminate them at this point. If the whole image is good and you want to outline the everything choose Select All from the Edit menu. Once you have made your selection go to the Special menu and choose [1] Set Region of Interest. 2. Now you have to decide the intensity of the rolling ball background subtraction you are going to make. Choose an image of your most heavily infected cells. Then using the Strait line Selection tool (Located in the Tools pallet on the left side of the screen: outlined in red FIG 2) draw a line selection across the largest inclusion in the Image and choose [2] Calculate Background subtraction from the Special menu. Follow the instructions and make a note of the rolling ball radius. You may want to check a number of images in this way, but dont worry it does not have to be too precise. If you make a stack out of a number of images of infected cells. 3. Depending largely on the size of the rolling ball this part of the macro will take some time. If you are processing a lot of images (unless you have a 604e or G3 Power Mac) it is time to do something else! The next version of Inclusion Counter will include an option to use the faster implementation of the 2D rolling ball algorithm which is about ten times faster. Now you know which colour channel to use and the size of the rolling ball radius you need run the appropriate macro from the following list: Grey Scale. Background Subtraction 24 Bit to Grey Scale. Background Subtraction Red Channel. Background Subtraction Green Channel. Background Subtraction Blue Channel. Background Subtraction Red and Green Channels. Background Subtraction Red and Blue Channels. Background Subtraction Green and Blue Channels. Background Subtraction follow the instruction and enter the rolling ball radius when requested. Your images will now be save as Subtracted.0001....... 4. Now choose macro [3] Tell me how to estimate particle size range! and follow the on screen instructions. 6. When adjusting the density slice in both [4] Positive and Negative Controls I . Analyse Particles, [5] Positive and Negative Controls II . Inclusion Size and [8] Background Subtracted Images. Automated Inclusion Enumeration you can allow some of the particulate back ground to be highlighted as this will be removed when the images are converted to binary images. You really have to repeatedly play with steps [4] and [5] until you are happy that you have got the best results possible. Make sure you record all the details like Density slice values and particle sizes for use in step [8]. 7. Set the scale of you images using [6] Set Scale part 1 and [7] Set Scale part 2. Make a note of the scale and go on to [8] Background Subtracted Images. Automated Inclusion Enumeration. 8. By now you should know the following values: Scale; Units; upper density slice setting; lower density slice setting; minimum particle size; and maximum particle size. You will be prompted to enter these values by [8] Background Subtracted Images. Automated Inclusion Enumeration. 9. Open the results generated by [8] Background Subtracted Images. Automated Inclusion Enumeration in a spread sheet that has a Parse command, e.g. Excel 3.0 onwards. This will arrange the results neatly into columns of the spread sheet for statistical analysis and display purposed. Manual Density Slice v1.0 1. Set the region of the images which you are interested in or ROI. Choose any of the area selection tools (FIG 1) and outline the area you want. If like me your camera is quite old and has a few bad lines at one edge you can eliminate them at this point. If the whole image is good and you want to outline the everything choose Select All from the Edit menu. Once you have made your selection go to the Special menu and choose [1] Set Region of Interest. 21. Now you have to decide the intensity of the rolling ball background subtraction you are going to make. Choose an image of your most heavily infected cells. Then using the Strait line Selection tool (Located in the Tools pallet on the left side of the screen FIG 2) draw a line selection across the largest inclusion in the Image and choose [2] Calculate Background subtraction from the Special menu. Follow the instructions and make a note of the rolling ball radius. You may want to check a number of images in this way, but dont worry it does not have to be too precise. If you make a stack out of a number of images of infected cells. 3. Depending largely on the size of the rolling ball this part of the macro will take some time. If you are processing a lot of images (unless you have a 604e or G3 iPower Mac) it is time to do something else! The next version of Inclusion Counter will include an option to use the faster implementation of the 2D rolling ball algorithm which is about ten times faster. Now you know which colour channel to use and the size of the rolling ball radius you need run the appropriate macro from the following list: Grey Scale. Background Subtraction 24 Bit to Grey Scale. Background Subtraction Red Channel. Background Subtraction Green Channel. Background Subtraction Blue Channel. Background Subtraction Red and Green Channels. Background Subtraction Red and Blue Channels. Background Subtraction Green and Blue Channels. Background Subtraction follow the instruction and enter the rolling ball radius when requested. Your images will now be save as Subtracted.0001....... 4. Depending on the type of staining you have used choose [3] Manual Density Slice Background Subtracted Images or [4] Manual Density Slice Background Subtracted Fluorescent Images. These macros will allow you to individually set the density slice of each image and save the results as binary images for further processing. You can allow some of the particulate back ground to be highlighted as this will be removed after the images are converted to binary images. 6. Now run macro [5] Tell me how to estimate particle size range! an follow the on screen instructions. 7. u [6] Positive and Negative Controls I . Analyse Particles and [7] Positive and Negative Controls II . Inclusion Size follow the on screen instructions. Again you really have to repeatedly play with steps [6] and [7] until you are happy that you have got the best results possible. It is a good idea to have to original image available for comparison at this point. Make sure you record all the details required for use in step [10]. 8. Set the scale of you images using [8] Set Scale part 1 and [9] Set Scale part 2. Then go on to [10] Binary Images. Automated Inclusion Enumeration. 9. By now you should know the following values: Scale; Units; minimum particle size; and maximum particle size. You will be prompted to enter these values by [10] Binary Images. Automated Inclusion Enumeration. 10. Open the results generated by [10] Background Subtracted Images. Automated Inclusion Enumeration in a spread sheet that has a Parse command, e.g. Excel 3.0 onwards. This will arrange the results neatly into columns of the spread sheet for statistical analysis and display purposed. No Background Subtraction v1.0 1. Set the region of the images which you are interested in or ROI. Choose any of the area selection tools (FIG 1)and outline the area you want. If like me your camera is quite old and has a few bad lines at one edge you can eliminate them at this point. If the whole image is good and you want to outline the everything choose Select All from the Edit menu. Once you have made your selection go to the Special menu and choose [1] Set Region of Interest. 2.e Depending on the type of staining you have used choose ..... Manual Density Slice or ..... Fluorescent Manual Density Slice. These macros will allow you to individually set the density slice of each image and save the results as binary images for further processing. You can allow some of the particulate back ground to be highlighted as this will be removed after the images are converted to binary images. 6. Now run macro [2] Tell me how to estimate particle size range! an follow the on screen instructions. 7. [3] Positive and Negative Controls II . Inclusion Size and [4] Positive and Negative Controls I . Analyse Particles follow the on screen instructions. Again you really have to repeatedly play with steps [3] and [4] until you are happy that you have got the best results possible. It is a good idea to have to original image available for comparison at this point. Make sure you record all the details required for use in step [7]. 8. Set the scale of you images using [5] Set Scale part 1 and [6] Set Scale part 2. Then go on to [7] Binary Images. Automated Inclusion Enumeration. 9. By now you should know the following values: Scale; Units; minimum particle size; and maximum particle size. You will be prompted to enter these values by [7] Binary Images. Automated Inclusion Enumeration. 10. Open the results generated by [7] Background Subtracted Images. Automated Inclusion Enumeration in a spread sheet that has a Parse command, e.g. Excel 3.0 onwards. This will arrange the results neatly into columns of the spread sheet for statistical analysis and display purposed. Photoshop Helper v1.0 1. Set the region of the images which you are interested in or ROI. Choose any of the area selection tools (FIG 1)and outline the area you want. If like me your camera is quite old and has a few bad lines at one edge you can eliminate them at this point. If the whole image is good and you want to outline the everything choose Select All from the Edit menu. Once you have made your selection go to the Special menu and choose [1] Set Region of Interest. 2. Set the scale of you images using [2] Set Scale part 1 and [3] Set Scale part 2. Then go on to 4. Process Photoshop Images. 3. Choose 4. Process Photoshop Images and enter the Scale and Units as prompted. 4. Open the results generated by [4] Process Photoshop Images in a spread sheet that has a Parse command, e.g. Excel 3.0 onwards. This will arrange the results neatly into columns of the spread sheet for statistical analysis and display purposed. 8. Photoshop 4.0 and Inclusion Counter You will find an Photoshop 4.0 action pallet located in the Inclusion Counter folder called Inclusion Counter Actions. To load this into Photoshop click on the Popup menu button in the actions pallet (outlined in red FIG 3) and choose Load Actions from the menu and open Inclusion Counter Actions from the Inclusion Counter folder supplied with inclusion counter. Inclusion Counter Actions will load into the actions pallet (FIG 4). Counting objects by manual outlining for quality control purposes. First you need to add a second layer press F4. The choose the lasso tool from the tools pallet FIG 5 (Red outline). Trace the outline of the object/Inclusion. Once you are happy press F6. This fills the outline in the second layer you created previously, leaving the original image untouched. Repeat this with all the inclusions/objects in the field. Alternatively you can use the wand tool FIG 5 (Blue outline). Click on the object of interest. If the outline does not cover the object press F5. Press F6 when you are happy with the selection. This is much faster than manually outlining. However, you may find that you need to use the manual tool on some objects. Ultimately, whilst outlining objects using the lasso tool is appallingly tedious, as long as optical density is not critical, it is by far the most accurate way of measuring the area of objects. Once you have outlined all the objects in the field press Shift_F1. Open your next image and repeat. Stop when you start feeling suicidal. Some image have taken me 40 minutes to process in this way! You will really appreciate Inclusion Counter once you have done 20 - 30 images in this way. I did 200 for one cell type and staining protocol, but >30 spread over a good range of inclusion/object numbers and sizes should be adequate statistically. Once you have done all the images you can stand, close all open image windows and set up a batch process (see below) using the action Delete Background and save as TIFF. Photoshop 4.0 & Photoshop Helper v1.0 Once you have batch processed the outlined images using Delete Background and save as TIFF you can use Inclusion Counter to count (See Photoshop He3lper v1.0 ) and measure the objects automatically. This is practically infallible since the images are now essentially Black & White binary images of objects with no background. However, make sure you use the same ROI settings as you used or intend to use for comparison, for obvious reasons. For security you can copy the ROI file you are using to somewhere out side the ROI Using Photoshop for file conversion Use the action Save as TIFF to convert all Photoshop readable formats, there are quite a few, to TIFF. Set up a Batch process as above. However, you can save over the original files in TIFF format, this will save you considerable Hard Disks space. Alternatively use GraphicConverter. Using Photoshop to overlay images counted in NIH-Image onto original images In NIH-Image choose Select All from the edit menu and copy the selection. Paste the contents into a fresh layer in the original images. Currently there is no way of automating this process. If I can think of one it will be implemented in future versions of Inclusion counter. Alternatively if you know of a Photoshop plug in that will do this let me know. Who knows maybe Photoshop 5.0 will be even better than 4.0 and support this kind of batch processing. Once you have completed all the images you want copy them to a separate folder and Batch process them using the action Clean up Counted Inclusions (2). NB This action will only work on the images to which you previous counted inclusions in using the lasso or wand technique, for naive images which you have counted with other Inclusion Counter macros use Clean up Counted Inclusions (1). The number in brackets refers to the layer into which the counted inclusions have been pasted. How to do batch processing in Photoshop 4.0 Choose the Popup menu button in the actions pallet (outlined in red FIG 3) and choose Batch from the menu. Select the folder with you images. Then choose a separate folder for the output and select the action you require. In most cases you must select a different folder to save you images in or they will save over your originals! However, if you are converting images from another format to Tiff you may what Photoshop to overwrite the originals. 10. Acknowledgements Many thanks to Dr Chris Coulon (The GAIA Group, ad1054@worldpath.net) for his intellectual input and eEncouragement, without which Inclusion Counter would not have been written. I would also like to thank the members of the NIH Image list server (nih-image@Soils.Umn.EDU) for their help and views relating to a number of issues. Particularly Mark Vivino, [2] Calculate Background subtraction is based largely on part of his cell colony macros. Obviously I must, as always, thank Wayne Rasband for his creation (NIH Image) and the U.S. National Institute of Health for their enlighten policy of making NIH Image Public Domain. I would also like to thank my Ph.D. supervisors Dr Gary Entrican and Dr Sarah E. M. Howie for their constant support. This work was sponsored by the Scottish Office Agriculture, Environment and Fisheries Department. 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